The expression levels of ghrelin, apolipoprotein B (apoB), exportin 4 (xpo4), peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (pin1), preproenkephalin 1 (penk1), and peroxiredoxin 2 (prdx2) genes were validated by qPCR. RNA samples were reverse-transcribed for 2 h at 37°C with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). qPCR was performed by using 1 µl of cDNA, 2× SYBR Green PCR Master Mix (Applied Biosystems), and 200 nM of forward and reverse primers. The reaction condition was followed: 10 min at 95°C, and 40 cycles of 15 sec at 95°C, 1 min at 60°C. Each assay was run on an Applied Biosystems 7300 Real-Time PCR system in triplicates. Fold changes were calculated using the comparative CT method. The primer set for each gene is followed: ghrelin forward, 5′-GCTGGAGATCAGGTTCAATGC-3′; ghrelin reverse, 5′-GTCCGTGGTTACTTGTCAGC-3′; apoB forward, 5′-TCTGCCTCTTACTACCCACTG-3′; apoB reverse, 5′-TGTCAACCAAAGACTTGTCCTC-3′; xpo4 forwards, 5′-AGATACCGCAGCTTCCTGAG-3′; xpo4 reverse, 5′- GTGGTCATCTCCGTGTTGTG-3′; pin1 forward, 5′-ATGGCGGACGAGGAGAAG-3′; pin1 reverse, 5′- CGAGACTGGCTGTGCTTC-3′; penk1 forward, 5′-CTTGGGTCCTGCCTCCTG-3′; penk1 reverse, 5′-GCAAGTGGCTCTCATCCTG-3′; prdx2 forward, 5′-CGACCATGCTGAGGACTTC-3′; prdx2 reverse, 5′- TCAACACGCCGTAATTCTGG-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5′-ACACCCACTCCTCCACCTTT-3′; GAPDH reverse, 5′-TAGCCAAATTCGTTGTCATACC-3′.
